In the past many scoffed at the idea that tissue culture might become a competitive process for propagating nursery crops. Today the tissue culture process is widely used worldwide for plant reproduction, and millions of cultivated plants are now produced through tissue culture. With appropriate techniques, tissue cultured plants are (1) genetically like the parent plant and (2) virus free. Thus, an outstanding plant can be a parent to millions of tissue cultured clones. A few examples of tissue cultured plants include orchids, kiwi fruit, strawberries, roses, and violets.
Tissue culture has the following potential applications: (1) production of natural products, (2) genetic improvement of crops, (3) production of disease-free plants, and (4) rapid multiplication. The last mentioned application probably has the greatest significance to a commercial propagator.
The usual tissue culture procedure is to place a particular part of the plant on top of a gel containing specific nutrients, and new plants are formed. A typical gel may contain agar. All operations are performed under sterile conditions. As the plants grow in a gel, various organic compounds are produced which, if not removed, slow the growth and eventually kill the plantlets. Usually the plantlets are removed to another container with the same or different gel, depending upon the stage of plantlet development.
An increasingly popular procedure is to avoid using a gel and to instead use a liquid medium. Instead of transplanting the plantlets to another container, the liquid nutrient solution is changed. Ideally, the nutrient solution is less than 0.5 mm deep at the level where the plantlet grows. If the plantlet is totally immersed in the nutrient solution, it will die. The container in which the plantlets are grown has a filter on the top so there is gas exchange between the outside air and gases inside the container.
A problem in micropropagation by tissue culture techniques using liquid media is to maintain a fixed thin liquid level for the plantlets while the liquid level inside the container is continuously reduced.
Many schemes have been used to solve this problem. For example, using a filter paper wick to transfer the solution has been tried, but the transfer rate is too low. Liquid sprays from above have been tried, but this is expensive and a commercial apparatus has not yet been introduced.
Recently, "membrane rafts" have been used. The idea is to have a membrane contact the liquid solution at a fixed level so that a thin film of solution is maintained above the membrane. The membrane promotes uptake of nutrients dissolved in the liquid media while it maintains the growing tissue culture in a relatively dry environment. Liquid media as opposed to gel procedure avoids exudate buildup and nutrient-depletion zones. This allows for greater media composition flexibility with the objective of making the entire growth process faster, easier, and more productive.
However, with the membrane raft system, everything works well until the plantlets' weight increases sufficiently that the raft sinks. One manufacturer has solved the problem by providing floats of various buoyancies, and as the plantlets increase in weight, one float is removed and a different float is placed under the membrane. The floats are available from one manufacturer in the following buoyancies: 0-5 grams, 0-10 grams, and 10-25 grams. As a further problem, if one corner of the raft has a relatively higher plantlet weight, that corner of the raft sinks lower than the other three corners. It is also rather difficult to increase the area of the raft/membrane system economically.
The present invention provides a process for improving micropropagation of plant material in liquid media. The process, among other improvements, overcomes the disadvantages of the membrane raft approach to micropropagation.